CN113355240A - Culture digestion equipment and culture digestion method for full-automatic culture of stem cells - Google Patents

Culture digestion equipment and culture digestion method for full-automatic culture of stem cells Download PDF

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Publication number
CN113355240A
CN113355240A CN202110753698.4A CN202110753698A CN113355240A CN 113355240 A CN113355240 A CN 113355240A CN 202110753698 A CN202110753698 A CN 202110753698A CN 113355240 A CN113355240 A CN 113355240A
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culture
box body
main box
bottom plate
digestion
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黄莹之
李捡平
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Ningbo Sinosat Biotechnology Co ltd
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Ningbo Sinosat Biotechnology Co ltd
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • C12M27/06—Stirrer or mobile mixing elements with horizontal or inclined stirrer shaft or axis
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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    • C12M39/00—Means for cleaning the apparatus or avoiding unwanted deposits of microorganisms
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control

Abstract

The invention provides a culture digestion device and a culture digestion method for full-automatic culture of stem cells, wherein the device comprises a main box body, a culture carrier storage platform, a sample inlet, a culture condition monitoring device, a spraying device, a filtering device and a processing device; the bottom plate of the main box body is provided with a switch device which is connected with the processing device and can open or close the bottom plate under the control of the control information; a first collection structure and a second collection structure are arranged below the switch device, and the first collection structure is used for collecting cell suspension and waste liquid after culture and digestion; the second collecting structure is used for collecting the honeycomb-shaped culture carriers. The culture digestion equipment and the culture digestion method for the full-automatic culture of the stem cells can realize full automation and no humanization of cell culture, can effectively reduce artificial components, are totally closed in the culture process, do not need to be operated in an open environment, and realize zero-pollution introduction; 3D culture is adopted, culture bottles and dishes are replaced, and the productivity is effectively improved.

Description

Culture digestion equipment and culture digestion method for full-automatic culture of stem cells
Technical Field
The invention relates to the technical field of stem cell culture, in particular to culture digestion equipment and a culture digestion method for full-automatic stem cell culture.
Background
With the continuous development and growth of the cell biotherapy industry, the progress of the cell culture technology provides important support for cells on new therapeutic technical means. In the process of cell culture, how to prevent cell pollution, how to realize large-scale production of cell culture and how to realize unmanned cell culture operation are a plurality of problems to be solved in the current cell culture technology.
The 2D to 3D culture is becoming a necessary direction for the development of cell culture technology, and the existing culture technology is that technicians inoculate samples in a class a environment by using cell bottles or cell dishes, and then transfer the samples to an incubator for cell culture, wherein the temperature in the incubator is set to be 37 ℃, and the carbon dioxide concentration is set to be 5%. Wherein relate to and operate in open environment, cause the pollution easily, whole culture system is operated by personnel, and the cell culture carrier is bottle or dish moreover, and a 175 blake bottle can cultivate 1 to 2 million cells at most, and a blake dish cultivates 5 million cells at most, in large-scale cultivation field, this production can't satisfy the market demand at all, and causes the pollution easily in the culture process.
With the improvement of living standard, the labor cost also gradually becomes the core cost in enterprise cost, so that the automation and the humanization also gradually become the development direction of new technology, and the cell culture can reduce the labor cost and solve the problem of pollution caused by the operation of technicians in an open environment while realizing the humanization. However, the existing cell culture field has no mature automatic unmanned cell culture equipment.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the culture digestion equipment and the culture digestion method for the full-automatic culture of the stem cells, which can realize unmanned and full-automatic operation and avoid the introduction of pollution to the greatest extent.
The invention adopts the following technical scheme for solving the technical problems:
the invention provides a culture and digestion device for full-automatic culture of stem cells, which comprises a main box body, a heating device and a control device, wherein the main box body is used for culturing and digesting the stem cells; the main box body is also provided with:
a culture carrier storage platform which can be opened or closed; the culture carrier storage platform can be communicated with the main box body or relatively sealed, and is used for adding the honeycomb culture carrier into the main box body when the culture carrier storage platform is communicated with the main box body;
the sample inlet can be opened or closed, is communicated with the main box body and is used for adding the seed cell and culture medium mixed solution into the main box body;
the culture condition monitoring device is communicated with the main box body and is used for monitoring and controlling the culture conditions in the main box body;
the spraying device is arranged in the main box body and is used for spraying and adding cleaning liquid, sterile water and digestive enzyme into the main box body;
the filtering device is detachably arranged at the lower end in the main box body and is used for filtering; and
processing means for issuing control information; the heating device, the culture carrier storage platform, the sample inlet, the culture condition monitoring device, the spraying device and the filtering device are all connected with the processing device and can be started to work or closed under the control of the control information;
the bottom plate of the main box body is provided with a switch device which is connected with the processing device and can open or close the bottom plate under the control of the processing device; a collecting device is arranged below the switch device and comprises a first collecting structure and a second collecting structure, the second collecting structure is connected with the lower end of the main box body, and the honeycomb-shaped culture carriers are discharged from the side wall of the second collecting structure; the first collecting structure is arranged at the lower end of the second collecting structure, and the switch device can also enable the first collecting structure to be communicated with the main box body under the control of the processing device so as to discharge cell suspension and waste liquid after culture and digestion.
Furthermore, the bottom of the culture carrier storage platform is provided with an automatic switch valve which is connected with the processing device and can be opened or closed under the control of the processing device.
Further, the culture condition monitoring device includes:
the carbon dioxide gas inlet channel is communicated with the main box body and is used for introducing carbon dioxide gas into the main box body and monitoring the concentration of the carbon dioxide in the main box body;
the temperature monitoring component comprises a temperature display screen, and the temperature display screen is arranged on the upper surface outside the main box body; the temperature monitoring component is used for monitoring the temperature in the main box body; and
the air balance component is communicated with the outside and the inside of the main box body, so that the outside can be communicated with the inside of the main box body; the air balance component is matched with the carbon dioxide inlet channel to adjust the concentration of the carbon dioxide in the main box body to be a preset concentration.
Further, the temperature monitoring component also comprises at least two temperature monitoring probes;
at least one temperature monitoring probe is arranged on the inner side wall of the main box body at a position corresponding to the liquid level of the culture solution and is used for monitoring the liquid level temperature; at least one temperature monitoring probe is arranged at the bottom of the inner side wall of the main box body and used for monitoring the temperature in the liquid surface.
Furthermore, a carbon dioxide concentration detection table is arranged on the carbon dioxide inlet channel;
the air balance component is provided with an air balance valve which can be opened or closed; the air balance part is internally provided with a filter membrane, and the filter membrane can pass gas but not liquid.
Furthermore, the side wall and the bottom plate of the main box body are divided into an inner layer and an outer layer, the inner layer is made of corrosion-resistant plastic materials, and the outer layer is made of metal materials; the heating device is arranged in the outer layer.
Furthermore, a mixing device is also arranged in the main box body; the mixing device comprises at least one mixing plate, and the mixing plate is parallel to a side plate of the main box body and is connected with the side plate of the main box body through a driving part; the mixing plate is movable within the main housing under the action of the driving means for mixing and activating the stem cells.
Furthermore, the bottom plate comprises two movable plates which are hinged with the middle part of the bottom plate to form a switch device; the two movable plates can be rotationally opened or closed relative to the middle part of the bottom plate under the control of the processing device.
Furthermore, the second collecting structure is a cavity with a semicircular section and comprises a curved bottom plate and two semicircular side plates; the two movable plates can rotate relative to the middle part of the bottom plate in the second collection structure, and the lower ends of the two movable plates are always contacted with the curved bottom plate when the two movable plates rotate;
two ends of the curved bottom plate are respectively provided with an opening which can be opened or closed and is used for discharging the honeycomb-shaped culture carrier; two ends of the curved bottom plate are respectively provided with a fixing part for fixing the movable plate relative to the curved bottom plate;
the first collecting structure comprises a vertical pipeline which is communicated with the lowest end of the curved bottom plate; the lower end of the vertical pipeline is provided with a first channel for discharging waste liquid and a second channel for discharging cell suspension in a branching manner; the first passage and the second passage are openable or closable.
The second aspect of the invention provides a culture digestion method for full-automatic stem cell culture, which adopts the culture digestion equipment for full-automatic stem cell culture as described above; the culture digestion method comprises two steps of cell culture and cell digestion; the cell culture comprises the following steps:
step one, installing and fixing a filtering device at the lower end in a main box body, and controlling a switch device to close a bottom plate;
adding a honeycomb culture carrier into the main box body through the culture carrier storage platform; adding a seed cell and culture medium mixed solution into the main box body through a sample inlet, and ensuring that the liquid level of the culture medium is highly covered with the honeycomb culture carrier;
regulating and monitoring culture condition information in the main box body through a culture condition monitoring device, and standing and culturing for 4-5 days;
the cell digestion comprises the following steps:
opening the bottom plate through the switch device, collecting waste liquid through the first collecting structure and the filtering device, and keeping the cell suspension in the main box body; after the waste liquid is discharged, the switch device closes the bottom plate;
step five, spraying cleaning fluid into the main box body by the spraying device, and repeating the step four to discharge waste liquid and cleaning fluid;
sixthly, adding digestive enzyme into the main box body by using a spraying device to digest and elute the cells for 3-5 minutes;
step seven, opening the bottom plate through the switch device, discharging the cell suspension through the first collection structure and the filtering device, and leaving the honeycomb culture carrier in the main box body; after the cell suspension is discharged, the switch device closes the bottom plate;
step eight, spraying cleaning fluid into the main box body by a spraying device, repeating the step six to elute the residual cells in the honeycomb culture carrier, and repeating the step seven to discharge cell suspension;
step nine, opening the bottom plate through the switch device, disassembling the filtering device, and discharging the honeycomb culture carriers through the second collecting structure; after the discharge is finished, the spraying device sprays high-pressure sterile water into the main box body to clean the main box body and remove waste liquid.
Furthermore, in the fourth step, the switch device is opened, the two movable plates rotate to be naturally vertical relative to the middle part of the bottom plate, the opening of the second collecting structure is closed, and the main box body is communicated with the first collecting structure; the first channel is opened and the second channel is closed, and then the cell culture supernatant is discharged as a waste liquid through the first channel.
Furthermore, in the seventh step, the switch device is opened, the two movable plates rotate to be naturally vertical relative to the middle part of the bottom plate, the opening of the second collecting structure is closed, and the main box body is communicated with the first collecting structure; the first channel is closed and the second channel is opened, and then the cell suspension is discharged through the second channel.
Further, in the ninth step, the opening and closing device is opened, the opening of the second collecting structure is opened, the two movable plates rotate relative to the middle of the bottom plate and are fixed through the fixing part, the main box body is not communicated with the first collecting structure, and the honeycomb-shaped culture carrier is discharged through the opening of the second collecting structure.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
1. the culture digestion equipment for the full-automatic culture of the stem cells, provided by the invention, is provided with the culture carrier storage platform and the sample inlet which can be opened by remote control and added with the honeycomb-shaped culture carrier and the mixed liquid of the seed cells and the culture medium, so that the manual inoculation of technicians is not needed, the automation of the culture process is realized, and the culture efficiency is higher; the main box body can contain 10L of culture medium, so that the capacity is higher;
2. the culture digestion equipment is provided with a culture condition monitoring device which can be remotely controlled, so that culture conditions such as gas, temperature and the like can be remotely adjusted in the culture process without manual operation of technicians; the controllable spraying device is also arranged, and the switching device and the collecting device which can be opened by remote control are arranged, so that the cell suspension can be discharged fully automatically after the culture is finished, the unmanned and full-closed culture process is realized, zero-pollution introduction is realized, the labor cost is reduced, and the cell culture quality is improved;
3. the culture carrier is a honeycomb spherical culture carrier made of gelatin, 3D culture can be realized by vertical growth of cells, about 80 hundred million cells can be cultured at one time, traditional cell culture carriers such as bottles and dishes are replaced, and the capacity is larger.
Drawings
FIG. 1 is a schematic diagram of an internal structure of a culture digestion apparatus for fully automatic stem cell culture according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the internal structure of a culture digestion device for the fully automatic culture of stem cells according to an embodiment of the present invention when waste liquid is discharged;
FIG. 3 is a schematic diagram of the internal structure of a culture digestion device for fully automatic stem cell culture according to an embodiment of the present invention when discharging a cell suspension;
FIG. 4 is a schematic diagram of the internal structure of the culture digestion device for the fully automatic culture of stem cells according to an embodiment of the present invention when discharging the cell culture carrier;
wherein the reference numerals are: 1-a main box body, 11-an outer layer, 12-an inner layer, 2-a culture carrier storage platform, 21-an automatic switch valve, 3-a sample inlet, 4-a culture condition monitoring device, 41-a carbon dioxide gas inlet channel, 42-a temperature monitoring part, 421-a temperature monitoring probe, 43-an air balancing part, 5-a spraying device, 6-a filtering device, 7-a switching device, 71-a movable plate, 8-a collecting device, 81-a first collecting structure, 811-a first channel, 812-a second channel, 82-a second collecting structure, 821-an opening, 822-a fixing part, 9-a mixing device, 91-a mixing plate and 92-a driving part.
Detailed Description
The present invention provides a culture digestion device for full-automatic stem cell culture, and the technical scheme of the present invention is further described in detail below with reference to the attached drawings to better understand the present invention, but not to limit the scope of the present invention.
Example 1
As shown in FIG. 1, the present embodiment provides a culture digestion device for full-automatic culture of stem cells, which comprises a main box 1, wherein the culture of stem cells is completed in the main box 1. The main box body 1 is also provided with a culture carrier storage platform 2, a sample inlet 3, a culture condition monitoring device 4, a spraying device 5, a filtering device 6 and a processing device.
The processing device is set as a PLC control module, comprises an instruction and data memory, an input unit, an output unit, a digital-analog conversion unit and the like, and is used for technicians to carry out remote operation and send out control information. The heating device, the culture carrier storage platform 2, the sample inlet 3, the culture condition monitoring device 4, the spraying device 5 and the filtering device 6 are all connected with the processing device and can be started to work or closed under the control of the control information. The PLC control module is a controller widely applied to the field of industrial control in the prior art, and a person skilled in the art can set a specific instruction according to requirements to operate the culture and digestion equipment.
The culture carrier storage platform 2 can be opened or closed, and honeycomb-shaped culture carriers can be stored in the culture carrier storage platform in advance; the culture carrier storage platform 2 can be communicated with the main box body 1 or relatively sealed, and is used for adding honeycomb culture carriers into the main box body 1 for cell culture during communication.
The bottom of the culture carrier storage platform 2 is provided with an automatic switch valve 21, and the automatic switch valve 21 is connected with the processing device and can be opened or closed under the control of the processing device. After the honeycomb-shaped culture carriers are stored in the culture carrier storage platform 2, the upper end of the culture carrier storage platform is provided with an inlet and an outlet which can be opened or closed, and the culture carrier storage platform can completely isolate air after being closed, so that pollution and mixed bacteria in the outside air are prevented from entering the main box body 1 from the culture carrier storage platform 2.
The sample inlet 3 can be opened or closed, and when being communicated with the main box body 1, is used for adding the seed cell and culture medium mixed solution into the main box body 1 for cell culture. The injection port 3 can be completely isolated from air after being closed, so that pollution such as bacteria introduced through the injection port 3 is avoided.
The culture condition monitoring device 4 includes a carbon dioxide gas intake passage 41, a temperature monitoring part 42, and an air balance part 43. The carbon dioxide gas inlet passage 41 communicates with the main tank 1 to introduce carbon dioxide gas into the main tank 1 and monitor the concentration of carbon dioxide in the main tank 1. The air balance part 43 is communicated with the outside and the inside of the main box body 1, so that the outside can be communicated with the inside of the main box body 1; the air balance member 43 adjusts the concentration of carbon dioxide in the main casing 1 to a predetermined concentration in cooperation with the carbon dioxide intake passage 41. The temperature balancing component 43 is an air balancing valve which can be opened or closed, and a filtering membrane is arranged in the temperature balancing component, the aperture of the filtering membrane is 0.22 μm, gas can pass through but liquid can not pass through, and the temperature balancing component can isolate the pollution of bacteria and the like in the outside air and prevent the bacteria and the like from entering the main box body 1. The carbon dioxide concentration in the main body 1 can be adjusted to 5% by opening or closing the air balance valve in the air balance part 43 and the carbon dioxide intake passage 41 by the operation of the processing means, thereby providing a good condition for cell culture.
The temperature monitoring part 42 comprises a temperature display screen which is arranged on the outer upper surface of the main box body 1; the temperature monitoring part 42 is used for monitoring and displaying the temperature in the main box 1. The temperature detection means 42 further comprises at least two temperature monitoring probes 421. Wherein, at least one temperature monitoring probe 421 is arranged on the inner side wall of the main box 1 at a position corresponding to the liquid level of the culture solution for monitoring the liquid level temperature; at least one temperature monitoring probe 421 is disposed at the bottom of the inner sidewall of the main tank 1 for monitoring the temperature in the liquid surface. The temperature display screen can respectively display the liquid surface temperature and the temperature in the liquid surface so as to provide observation reference for technicians and further judge whether the cell culture is normally carried out.
And the spraying device 5 is arranged in the main box body 1 and is used for spraying and adding cleaning liquid, sterile water and digestive enzyme into the main box body 1. Spray set 5 is the high pressure water spray valve, and sets up to telescopic ball shower nozzle, can 360 degrees three-dimensional spraying, when not needing to spray the during operation, spray set 5 retracts in the roof of main tank 1, when needing to spray the during operation, can stretch out under processing apparatus's control and spray the work.
The heating device is arranged in the side wall of the main box body 1 and used for heating the main box body 1 to maintain a preset temperature so as to meet the temperature condition required by cell culture. The side wall and the bottom plate of the main box body 1 are divided into an inner layer 12 and an outer layer 11, and the inner layer 12 is made of corrosion-resistant plastic, preferably PVC. The outer layer 11 is made of metal, preferably stainless steel; the heating device is arranged in the outer layer 11 and comprises a heating wire, an insulating layer and the like, the heating wire is connected with a power supply, the temperature of the main box body 1 is maintained to be 37 ℃ by heating the outer layer 11 of the main box body 1, and the temperature condition required by cell culture is met. The heating wire can be coiled in a circuitous way and arranged in the outer layer 11 for heating, and the heating effect is better and uniform.
The filtering device 6 is detachably installed at the lower end of the inside of the main case 1 for filtering, and the filtering mesh size of the filtering device 6 is 70 μm, which allows the liquid and the digested culture solution to pass through, but the culture carrier does not pass through, for discharging the culture solution, the waste liquid, and the culture carrier in the main case 1 in several times. The filtering device 6 is inserted into the main box body 1 through the gaps at the bottoms of the two side plates of the main box body 1, and the gaps can keep sealed after the filtering device 6 is inserted. The filtering device 6 can also be provided with a connecting driving part, and the automatic insertion and removal can be carried out through the power of the driving part, so that the automatic installation and the disassembly of the filtering device 6 are realized.
The main box body 1 is also internally provided with a hybrid device 9; the mixing device 9 comprises at least one mixing plate 91, the mixing plate 91 is parallel to a side plate of the main box body 1 and is connected with the side plate of the main box body 1 through a driving part 92; the mixing plate 91 is movable within the main housing 1 by the driving member 92 to mix and digest the stem cells. The driving means 92 can move the mixing plate 91 in the main body 1 in a direction perpendicular to the side plates while gradually moving away from or close to the side plates and always keeping parallel to the side plates. The mixing plates 91 are generally provided in two, and are symmetrically and oppositely disposed in the main chamber 1, and the cells in the main chamber 1 can be sufficiently digested and eluted by the movement of the mixing plates 91.
A switch device 7 is arranged on the bottom plate of the main box body 1, is connected with the processing device and can open or close the bottom plate under the control of the control information; the bottom plate comprises two movable plates 71, and the two movable plates 71 are hinged with the middle part of the bottom plate to form a switch device 7; the two movable plates 71 can be rotated to open or close relative to the middle of the bottom plate under the control of the processing device.
A collecting device 8 is arranged below the switch device 7, the collecting device 8 comprises a first collecting structure 81 and a second collecting structure 82, the second collecting structure 82 is connected with the lower end of the main box body 1, and the honeycomb-shaped culture carriers are discharged through the side wall of the second collecting structure 82; the first collection structure 81 is arranged at the lower end of the second collection structure 82, and the switch device 7 can also make the first collection structure 81 communicated with the main box 1 under the control of the processing device so as to discharge the cell suspension and waste liquid after culture digestion.
The second collecting structure 82 is a cavity with a semicircular section and comprises a curved bottom plate and two semicircular side plates; the two movable plates 71 can rotate in the second collecting structure 82 relative to the middle of the bottom plate, and the lower ends of the two movable plates always contact with the curved bottom plate during rotation.
As shown in fig. 4, both ends of the curved bottom plate are respectively provided with an opening 821 that can be opened or closed for discharging the honeycomb-shaped culture carrier; the two ends of the curved bottom plate are respectively provided with a fixing member 822 for fixing the movable plate 71 relative to the curved bottom plate. The fixed component 822 is a telescopic fixing column disposed on the curved bottom plate, and when the movable plate 71 slides to the fixed component 822 along the curved bottom plate, the movable plate can be blocked and fixed by the fixed component 822, and when the fixed component 822 retracts, the rotation of the movable plate 71 is not affected.
The first collecting structure 81 comprises a vertical pipe which is communicated with the lowest end of the curved bottom plate; the vertical pipe is branched at the lower end thereof to be provided with a first channel 811 for discharging waste liquid and a second channel 812 for discharging cell suspension; the first channel 811 and the second channel 812 are openable or closable. Valves are arranged in the first channel 811 and the second channel 812, and are connected with the processing device and can be opened or closed according to control information.
The switch device 7 and the collecting device 8 can be matched with each other to automatically discharge waste liquid, cell suspension and culture carriers respectively, and are matched with the mixing device 9 and the spraying device 5 to digest and elute cell culture liquid, so that waste caused by the fact that cells in part of carriers cannot be eluted can be avoided, and the productivity is improved to the greatest extent.
The culture digestion device provided by the embodiment realizes the automatic culture digestion step of the cells, and a person skilled in the art can also arrange a second channel 812 to connect the centrifugal device and the cryopreservation device, so as to complete the subsequent work of cell culture.
Example 2
As shown in fig. 1 to 4, the present embodiment provides a culture digestion method for fully automatically culturing stem cells, which uses the culture digestion apparatus provided in embodiment 1. The culture digestion method comprises two steps of cell culture and cell digestion; the cell culture comprises the following steps:
step one, installing and fixing a filtering device 6 at the lower end in a main box body 1, keeping the main box body 1 sealed, and controlling a switch device 7 to close a bottom plate;
secondly, adding a honeycomb culture carrier into the main box body 1 through the culture carrier storage platform 2; adding a seed cell and culture medium mixed solution into the main box body 1 through the sample inlet 3, and ensuring that the liquid level of the culture medium is highly covered with the honeycomb culture carrier;
and step three, regulating and monitoring the culture condition information in the main box body 1 through a culture condition monitoring device 4, and performing static culture for 4-5 days.
Specifically, the third step includes adjusting the concentration of carbon dioxide in the main tank 1 to 5% through the carbon dioxide intake passage 41 and the air balancing part 43, maintaining the temperature of the main tank 1 heated by the heating means to 37 ℃, and continuously monitoring and displaying the temperature in the liquid surface and the liquid surface in the main tank 1 through the temperature monitoring part 42.
The cell digestion comprises the following steps:
step four, opening a bottom plate of the main box body 1 through the switch device 7, discharging waste liquid through the first collecting structure 81 and the filtering device 6, and keeping the cell suspension in the main box body 1; after the waste liquid is discharged, the switch device 7 closes the bottom plate;
step five, spraying cleaning solution into the main box body 1 by the spraying device 5, and repeating the step four to discharge waste liquid and cleaning solution;
sixthly, adding digestive enzyme into the main box body 1 by the spraying device 5 to digest and elute the cells for 3-5 minutes;
step seven, opening the bottom plate through the switch device 7, discharging the cell suspension through the first collecting structure 81 and the filtering device 6, and leaving the honeycomb culture carrier in the main box body 1; after the cell suspension is completely discharged, the switch device 7 closes the bottom plate under the control;
step eight, spraying cleaning fluid into the main box body 1 by the spraying device 5, repeating the step six to elute the residual cells in the honeycomb culture carrier, and repeating the step seven to discharge cell suspension;
step nine, opening the bottom plate through the switch device 7, detaching the filter device 6, and discharging the honeycomb culture carriers through the second collection structure 82; after the waste liquid is completely discharged, the spraying device 5 sprays high-pressure sterile water into the main box body 1 to clean the main box body 1 and discharge the waste liquid.
As shown in FIG. 2, in the present embodiment, in the fourth step, the switch device 7 is opened, the two movable plates 71 are rotated to be vertical to the middle of the bottom plate, and then the cell culture supernatant is discharged as waste liquid through the first collecting structure 81, and the undigested cells and culture medium are not discharged due to the arrangement of the filtering device 6. Specifically, the first channel 811 is now open and the second channel 812 is closed.
In the sixth step, the cultured cells and the culture medium in the main box body 1 are also mixed and stirred through the mixing plate 91 of the mixing device 9, and the cells in the main box body 1 are sufficiently digested and eluted in cooperation with digestive enzymes.
In step seven, as shown in fig. 3, the switch device 7 is opened, the two movable plates 71 are rotated to be vertical to the middle of the bottom plate, and the digested cultured cells can pass through the filter device 6 and then be discharged through the first collection structure 81. At this point the first channel 811 is closed and the second channel 812 is open and the cell suspension is drained through the second channel 812.
In the ninth step, as shown in fig. 4, the switch device 7 is turned on, the two movable plates 71 rotate relative to the middle of the bottom plate, the fixing part 822 extends out, the movable plates 71 are fixed by the fixing part 822, and the opening 821 is controlled to open, so that the honeycomb-shaped culture carrier can slide along the movable plates 71 to the opening 821 and be discharged, and the branched discharge and discharge of the culture carrier and the culture solution are realized.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications or alterations to this practice will occur to those skilled in the art and are intended to be within the scope of this invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims (10)

1. A culture and digestion device for full-automatic culture of stem cells is characterized by comprising a main box body (1) for culturing and digesting the stem cells, wherein a heating device is arranged in the side wall of the main box body (1) for heating the main box body (1) and maintaining the interior of the main box body at a preset temperature; the main box body (1) is also provided with:
a culture carrier storage platform (2) which can be opened or closed; the culture carrier storage platform (2) can be communicated with the main box body (1) or is relatively closed, and is used for adding honeycomb culture carriers into the main box body (1) during communication;
the sample inlet (3) can be opened or closed, is communicated with the main box body (1) and is used for adding seed cell and culture medium mixed liquid into the main box body (1);
the culture condition monitoring device (4) is communicated with the interior of the main box body (1) and is used for monitoring and controlling the culture condition in the main box body (1);
the spraying device (5) is arranged in the main box body (1) and is used for spraying and adding cleaning solution, sterile water and digestive enzyme into the main box body (1);
the filtering device (6) is detachably arranged at the lower end in the main box body (1) and is used for filtering; and
processing means for issuing control information; the heating device, the culture carrier storage platform (2), the sample inlet (3), the culture condition monitoring device (4), the spraying device (5) and the filtering device (6) are all connected with the processing device and can be started to work or closed under the control of the control information;
a switch device (7) is arranged on the bottom plate of the main box body (1), is connected with the processing device and can open or close the bottom plate under the control of the processing device; a collecting device (8) is arranged below the switch device (7), the collecting device (8) comprises a first collecting structure (81) and a second collecting structure (82), the second collecting structure (82) is connected with the lower end of the main box body (1), and the honeycomb-shaped culture carriers are discharged through the side wall of the second collecting structure; a first collection structure (81) is arranged at the lower end of the second collection structure (82), and the switch device (7) can also enable the first collection structure (81) to be communicated with the main box body (1) under the control of the processing device so as to discharge cell suspension and waste liquid after culture and digestion.
2. The culture digestion apparatus for the fully automatic culture of stem cells according to claim 1, characterized in that the bottom of the culture carrier storage platform (2) is provided with an automatic switch valve (21), and the automatic switch valve (21) is connected with the processing device and can be opened or closed under the control of the processing device.
3. Culture digestion apparatus for the fully automated culture of stem cells according to claim 1, characterized in that the culture condition monitoring device (4) comprises:
the carbon dioxide gas inlet channel (41) is communicated with the main box body (1) and is used for introducing carbon dioxide gas into the main box body (1) and monitoring the concentration of the carbon dioxide in the main box body (1);
the temperature monitoring component (42) comprises a temperature display screen, and the temperature display screen is arranged on the outer upper surface of the main box body (1); the temperature monitoring part (42) is used for monitoring and displaying the temperature in the main box body (1); and
an air balance member (43) which communicates the outside with the inside of the main case (1) and allows the outside to communicate with the inside of the main case (1); the air balance part (43) is matched with the carbon dioxide air inlet channel (41) to adjust the concentration of carbon dioxide in the main box body (1) to be a preset concentration.
4. The culture digestion apparatus for the full-automatic culture of stem cells according to claim 3, wherein the temperature detection part (42) further comprises at least two temperature monitoring probes (421);
the at least one temperature monitoring probe (421) is arranged on the inner side wall of the main box body (1) at a position corresponding to the liquid level of the culture solution and is used for monitoring the liquid level temperature; at least one temperature monitoring probe (421) is arranged at the bottom of the inner side wall of the main box body (1) and used for monitoring the temperature in the liquid surface.
5. The culture digestion apparatus for the full-automatic culture of stem cells according to claim 3, wherein the carbon dioxide inlet channel (41) is provided with a carbon dioxide concentration detection table;
the air balance component (43) is provided with an air balance valve which can be opened or closed; a filter membrane is arranged in the air balance part (43), and the filter membrane can pass gas but not liquid.
6. The culture digestion apparatus for the full-automatic culture of stem cells according to claim 1, wherein the side wall and the bottom plate of the main box body (1) are divided into an inner layer (12) and an outer layer (11), the inner layer (12) is made of corrosion-resistant plastic material, and the outer layer (11) is made of metal material; the heating device is arranged in the outer layer (11).
7. The culture digestion apparatus for the full-automatic culture of stem cells according to claim 1, characterized in that a mixing device (9) is further arranged in the main box body (1); the mixing device (9) comprises at least one mixing plate (91), the mixing plate (91) is parallel to a side plate of the main box body (1) and is connected with the side plate of the main box body (1) through a driving part (92); the mixing plate (91) is movable within the main housing (1) by the driving means (92) for mixing and activating the stem cells.
8. The culture digestion apparatus for the fully automatic culture of stem cells according to claim 1, characterized in that the bottom plate comprises two movable plates (71), the two movable plates (71) are hinged with the middle of the bottom plate to form the switch device (7); the two movable plates (71) can be rotationally opened or closed relative to the middle part of the bottom plate under the control of the processing device.
9. The culture digestion apparatus for the full-automatic culture of stem cells according to claim 8, wherein the second collection structure (82) is a cavity with a semicircular cross section, and comprises a curved bottom plate and two semicircular side plates; the two movable plates (71) can rotate relative to the middle part of the bottom plate in the second collection structure (82), and the lower ends of the two movable plates are always in contact with the curved bottom plate during rotation;
both ends of the curved bottom plate are respectively provided with an opening (821) which can be opened or closed and is used for discharging the honeycomb culture carrier; two ends of the curved bottom plate are respectively provided with a fixing part (822) for fixing the movable plate (71) relative to the curved bottom plate;
the first collecting structure (81) comprises a vertical pipeline which is communicated with the lowest end of the curved bottom plate; the vertical pipe is branched at the lower end thereof to be provided with a first channel (811) for discharging the waste liquid and a second channel (812) for discharging the cell suspension; the first channel (811) and the second channel (812) are openable or closable.
10. A culture digestion method for full-automatic culture of stem cells, which is characterized by adopting the culture digestion equipment for full-automatic culture of stem cells according to any one of claims 1-9; the culture method comprises two steps of cell culture and cell digestion; the cell culture comprises the following steps:
step one, the filtering device (6) is fixedly arranged at the lower end in the main box body (1), and the switch device (7) is controlled to close the bottom plate;
secondly, adding the honeycomb-shaped culture carrier into the main box body (1) through the culture carrier storage platform (2); seed cell and culture medium mixed liquid is added into the main box body (1) through the sample inlet (3), and the liquid level of the culture medium is ensured to cover the honeycomb culture carrier;
regulating and monitoring culture condition information in the main box body (1) through the culture condition monitoring device (4), and standing and culturing for 4-5 days;
the cell digestion comprises the following steps:
step four, after the culture is finished, opening the bottom plate through the switch device (7), discharging the waste liquid through the first collecting structure (81) and the filtering device (6), and keeping the cell suspension in the main box body (1); after the waste liquid is discharged, the switch device (7) closes the bottom plate;
fifthly, spraying cleaning liquid into the main box body (1) by the spraying device (5), and repeating the fourth step to discharge waste liquid and cleaning liquid;
sixthly, adding digestive enzyme into the main box body (1) by the spraying device (5) to digest and elute cells for 3-5 minutes;
seventhly, opening the bottom plate through the switch device (7), and collecting the cell suspension through the first collecting structure (81) and the filtering device (6), wherein the honeycomb-shaped culture carrier is left in the main box body (1); after the cell suspension is discharged, the switch device (7) closes the bottom plate;
step eight, spraying cleaning fluid into the main box body (1) by the spraying device (5), repeating the step six to elute the residual cells in the honeycomb culture carrier, and repeating the step seven to discharge the cell suspension;
ninthly, opening the bottom plate through the switch device (7), detaching the filtering device (6), and discharging the honeycomb-shaped culture carriers through the second collecting structure (82); after the discharge is finished, the spraying device (5) sprays high-pressure sterile water into the main box body (1) to clean the main box body (1) and remove waste liquid.
CN202110753698.4A 2021-07-02 2021-07-02 Culture digestion equipment and culture digestion method for full-automatic culture of stem cells Pending CN113355240A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110753698.4A CN113355240A (en) 2021-07-02 2021-07-02 Culture digestion equipment and culture digestion method for full-automatic culture of stem cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110753698.4A CN113355240A (en) 2021-07-02 2021-07-02 Culture digestion equipment and culture digestion method for full-automatic culture of stem cells

Publications (1)

Publication Number Publication Date
CN113355240A true CN113355240A (en) 2021-09-07

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Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110753698.4A Pending CN113355240A (en) 2021-07-02 2021-07-02 Culture digestion equipment and culture digestion method for full-automatic culture of stem cells

Country Status (1)

Country Link
CN (1) CN113355240A (en)

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